The long-term[unreadable] objective of these studies are to determine how an enteropathogenic[unreadable] bacterium is able to enter within host cells and to evaluate the role[unreadable] of mammalian cell receptors in promoting uptake. As a model system,[unreadable] Yersinia pseudotuberculosis is being studied in order to gain detailed[unreadable] information on the function of bacterial- and host-encoded[unreadable] internalization factors. Specifically, Y. pseudotuberculosis invasin-[unreadable] integrin interaction will be studied, and host-encoded factors that[unreadable] modulate receptor-mediated bacterial internalization will be identified.[unreadable] Uptake promoted by invasin depends upon high affinity binding to its[unreadable] receptors, regulated by the concentration of receptor available to[unreadable] participate in uptake. To further investigate the molecular mechanism[unreadable] of uptake, the following experiments will be performed: 1) A model for[unreadable] the binding of a single invasin molecule to multiple host receptors will[unreadable] be tested, by analyzing the subunit structure of invasin and determining[unreadable] the stoichiometry of invasin-receptor interaction; 2) the region of the[unreadable] integrin heterodimer involved in invasin binding will be determined by[unreadable] performing two novel mutant selection for altered receptor interaction;[unreadable] 3)the role in bacterial uptake of the beta-1 integrin will be[unreadable] investigated by analyzing the binding of mammalian cell cytoplasmic[unreadable] components to hybrid protein harboring this domain; 4) functional[unreadable] studies on the role of mammalian cytoplasmic components will be[unreadable] performed using a newly-developed perforated cell assay, allowing[unreadable] evaluation of the biological roles of factors identified in other Aims;[unreadable] and5) the role of invasin in intestinal infections will be analyzed,[unreadable] using invasin mutations resulting in partially functional proteins.[unreadable] Bacterial uptake by host cells is a common step in establishing disease[unreadable] by a number of bacterial pathogens. Investigation of this process will[unreadable] result in important information on how enteric diseases are initiated,[unreadable] and provide a potential source for the development of new chemotherapies[unreadable] that block this step in the infection process. In addition,[unreadable] identification of the components that allow a simple organism to enter[unreadable] an animal cell could result in new techniques to introduce therapeutic[unreadable] agents that would otherwise not be able to enter the host cell.[unreadable]